灰葡萄孢犬尿氨酸单氧酶基因BcKMO调控病菌致病力的机制分析

doi:10.3864/j.issn.0578-1752.2014.16.005

摘要/Abstract

摘要： 【目的】揭示灰葡萄孢犬尿氨酸单氧酶基因BcKMO（kynurenine 3-monooxygenase）调控病菌致病力的机制，阐明灰葡萄孢致病的分子机理。【方法】利用DNS和Hoffman方法，对BcKMO的T-DNA插入突变体BCG183和回复突变体（BCG183/BcKMO）的多聚半乳糖醛酸酶（PMG）、果胶酶（PG）、纤维素酶（Cx）、多聚半乳糖醛酸反式消除酶（PGTE）和果胶甲基反式消除酶（PMTE）的活性进行分析；提取灰葡萄孢野生型和BCG183、BCG183/BcKMO突变体的粗毒素并进行生物活性测定；利用溴百里酚蓝显色试验，对突变体BCG183和BCG183/BcKMO的产酸能力进行分析；利用洋葱表皮穿透试验，检测突变体BCG183和BCG183/BcKMO的穿透能力；利用real-time PCR技术，检测突变体BCG183和BCG183/BcKMO中已知致病相关基因腺苷酸环化酶编码基因Bac、异源三聚体G-蛋白G?亚基基因Bcg2和Bcg3、蛋白激酶调节亚基基因PkaR、MAP激酶编码基因Bmp1和Sak1、MAP激酶Bmp3和BcSak1下游的靶基因Bcreg1、TCHK-MAPK信号途径基因Bos1、亲环蛋白A编码基因Bcp1、小G蛋白基因Ras2、多聚半乳糖醛酸酶基因Bcpg1和超氧化物歧化酶基因Sod1的表达水平。【结果】突变体BCG183中的PMG和PG的酶活性较野生型和回复突变体明显增强，CX、PGTE和PMTE的酶活性与野生型和回复突变体没有明显差别；突变体BCG183的毒素活性较野生型和回复突变体明显增强；突变体BCG183的产酸能力较野生型和回复突变体明显减弱；突变体BCG183能够穿透洋葱表皮，在培养基上形成菌落，与野生型和回复突变体没有明显差别；突变体BCG183中已知致病相关基因Bac、Bcg2、Bcg3、PkaR、Bmp1、Sak1、Bcreg1、Bos1、Bcp1、Ras2、Bcpg1和Sod1的表达水平明显强于野生型和回复突变体。【结论】灰葡萄孢BcKMO通过调控病菌的胞壁降解酶活性、毒素活性、产酸能力、致病相关基因的表达而影响病菌的致病力。

关键词: 灰葡萄孢 , BcKMO , 突变体 , 致病力

Abstract: 【Objective】The objective of this study is to reveal the molecular mechanism of kynurenine 3-monooxygenase gene BcKMO in regulating pathogenicity of Botrytis cinerea and lay a foundation for clarifing the pathogenic mechanism of B. cinerea in the future.【Method】The activities of polymethylgalacturonase (PMG), endopolygalacturonase (PG), cellulase (CX), polygalacturonic acid transeliminase (PGTE) and pectin methyl transelimination enzyme (PMTE) were analyzed in the wild-type strain (WT), the BcKMO gene ATMT mutant (BCG183) and gene complement mutant (BCG183/BcKMO) by DNS and Hoffman methods. The toxin was isolated from WT, BCG183 and BCG183/BcKMO and the activity of toxin was analyzed. Acid production assays was performed on PDA medium with bromothymol blue. Penetrability of WT, BCG183 and BCG183/BcKMO was detected with onion epidermis spread on PDA medium. Real-time PCR was used to measure the transcription levels of pathogenicity-related genes, e.g., Bac, Bcg2, Bcg3, PkaR, Bmp1, Sak1, Bcreg1, Bos1, Bcp1, Ras2, Bcpg1, and Sod1 in WT, BCG183 and BCG183/BcKMO.【Result】The activity of PMG and PG in mutant BCG183 were significantly higher than that of WT and BCG183/BcKMO. The CX, PGTE and PMTE activities were no significant difference while compared with WT and BCG183/BcKMO. The toxin activity of mutant BCG183 was significantly higher than that of WT and BCG183/BcKMO. The acid production of the mutant BCG183 significantly decreased. The penetrability of mutant BCG183 appeared no significant difference compared to WT and BCG183/BcKMO. The expression level of pathogenicity-related genes, i.e., Bac, Bcg2, Bcg3, PkaR, Bmp1, Sak1, Bcreg1, Bos1, Bcp1, Ras2, Bcpg1, and Sod1, was obviously up-regulated in mutant BCG183.【Conclusion】The BcKMO gene is involved in regulating cell wall degradation enzyme activity, toxin activity, acid production, penetrability, and the expression of pathogenicity- related genes in B. cinerea.

Key words: Botrytis cinerea , BcKMO , mutants , pathogenicity

李培芬, 赵福鑫, 董丽萍, 郑会欣, 赵斌, 韩建民, 邢继红, 董金皋. 灰葡萄孢犬尿氨酸单氧酶基因BcKMO调控病菌致病力的机制分析[J]. 中国农业科学, 2014, 47(16): 3167-3173.

LI Pei-Fen, ZHAO Fu-Xin, DONG Li-Ping, ZHENG Hui-Xin, ZHAO Bin, HAN Jian-Min, XING Ji-Hong, DONG Jin-Gao. Mechanism Analysis of Kynurenine 3-monooxygenase Gene BcKMO in Regulation of Pathogenicity in Botrytis cinerea[J]. Scientia Agricultura Sinica, 2014, 47(16): 3167-3173.

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